Autoimmune diseases are characterized by an abnormal immune response (involving either immune system cells or antibodies) directed against normal autologous (self) tissues. Autoimmune diseases afflict huge numbers of individuals throughout the world. A common characteristic of several autoimmune diseases is the presence of one or more types of antinuclear antibodies (ANA) in the bodily fluids of patients suffering from the disease. ANA's are autoantibodies directed against antigens in the nucleus and cytoplasm of a person's own cells. It is thought that these antibodies are responsible for the tissue injuries associated with autoimmune disease. Thus, the detection of ANA's can be of significant clinical or diagnostic value.
Antinuclear antibodies are prominent in the biological fluids of patients suffering from disorders such as Systemic Lupus Erythematosus (SLE), Systemic Sclerosis, Mixed Connective Tissue Disease (MCTD), and Sjogren's Syndrome (Tan, E. M., 1989; Craft et al., 1993). Immunofluorescent tests using cultured human cell substrates are useful in determining the presence of ANA's in human sera (McDuffie et al., 1987) but do not provide information regarding the specificity of the antibodies. The disease specificity of well characterized antinuclear antibodies is an important diagnostic aid when interpreted in conjunction with the patient's clinical condition. It can also be used to partition patients into clinical subsets, and in some instances the presence or titer of a specific ANA correlates with the patient's prognosis (Reichlin, M., 1993). Currently, the determination of antinuclear antibody specificity requires considerable additional testing.
The use of indirect immunofluorescence (IF) for the detection of ANA's dates back to 1957. This method is still the standard method used to screen for the presence of ANAs. Typically, the patient's serum is diluted 1:40 in a buffer solution and allowed to react with human cells that have been fixed on a glass slide. If there are antibodies in the patient's serum that are immunoreactive with antigen components associated with the cell, they will bind to the cells and form an antigen-antibody complex. After washing to remove any unbound material, the presence of antigen-antibody complexes are detected using an anti-human antibody labeled with a fluorescent indicator. The presence of a fluorescent signal is detected by viewing the cells under a fluorescent microscope. The fluorescent signal is transient and will disappear within a few hours or days. Sera which test positive for ANA can then be titered out to an endpoint (i.e., diluted 1:80, 1:160, etc., and reacted with cells as before until a fluorescent signal can no longer be detected under the microscope). Disease severity, and in some cases response to therapy, is measured by increases and decreases in the ANA titer in a patient.
In addition to determining the presence or absence of fluorescent signal, detection of certain patterns of fluorescence also provides useful information in diagnosing the specific disease that may be afflicting a patient. For example, a homogeneous fluorescent nuclear signal (i.e., an even staining of the entire nucleus within the cell) is indicative of SLE, while a speckled pattern (i.e., a fine or granular-appearing staining of the nucleus) can indicate Scleroderma, MCTD, Sjogren's Syndrome or Raynaud's Syndrome. The staining pattern is due to the reactivity of the patients' antibodies to specific nuclear or cytoplasmic components.
Unfortunately, the IF method is not strictly diagnostic of specific autoimmune disease because of the substantial overlap of the fluorescent patterns exhibited by a number of autoimmune diseases. Also, if the sample is not titered appropriately, masking of fluorescent patterns can occur. Furthermore, even if a pattern is seen that is suggestive of a specific autoimmune disease, extensive confirmatory testing with purified antigens such as Sm, Scl-70, Ro, La, RNP and double stranded DNA, using assays such as EIA's, immunodiffusion or hemagglutination is necessary before one can be reasonably sure of a diagnosis. Thus, IF is a tedious method which does not generate a permanent record, involves multiple assays, is time-consuming and labor intensive, and requires considerable expertise in the interpretation of results.
Western blots (immunoblots) have been used to detect certain antinuclear antibody specificities (Francoeur, A. M., 1989). The use of immunoblots for the detection of specific antigens recognized by ANAs provides several advantages over IF, immunodiffusion, immunoprecipitation and EIA methods including a) more complete information can be obtained about the number and molecular size of antigen, b) large antigen complexes are separated into their component antigenic polypeptides yielding very specific ANA reactivities, and c) immunoblotting is extremely sensitive and reproducible (Habets, W. J., et al., 1983). The antigen specificity of the antibody can be determined by comparison of the sample reactivity with control markers on the blot. However, the Western blot method cannot be used to detect antibodies that are immunoreactive with double-stranded DNA (dsDNA) since membranes that bind proteins do not bind DNA very well unless the transfer occurs under alkaline conditions; unfortunately, dsDNA becomes single stranded under these conditions. And since antibodies that bind single-stranded DNA (ssDNA) are present in both autoimmune and non-autoimmune diseases, a positive ssDNA immunoblot result has little clinical significance.
In contrast, the presence of antibodies to dsDNA has both diagnostic and therapeutic importance (Harmon, C. E., 1985). Moreover, the presence of antibodies to dsDNA and to an RNA-protein complex called Sm are serologic criteria used in the classification of SLE. These and other ANA specificities often appear concomitantly in SLE. In addition, increases or decreases in the end-point titer of ANA's to dsDNA is correlated with, and used in monitoring, the clinical progression of SLE in patients suffering from the disease.
The inability to test for both antibodies to protein and double-stranded DNA antigen using Western blotting techniques requires that a separate assay for antibodies to dsDNA be conducted. This results in additional cost and manpower, as well as increasing the risk of errors. Thus, there remains a need for a single assay that is sensitive, simple to use, and which provides specific ANA results to aid in diagnosis and monitoring of autoimmune diseases.